Genetic therapies to lower cholesterol

VPH-06130; No of Pages 5 Vascular Pharmacology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Vascular Pharmacology

Review

2Q1

Genetic therapies to lower cholesterol

3Q2

Bernard Khoo ⁎

4Q3

Endocrinology, Division of Medicine, University College London, London, United Kingdom

5

a r t i c l e

6 7 8 9 10

Article history: Received 29 August 2014 Received in revised form 5 December 2014 Accepted 16 December 2014 Available online xxxx

11 Q5 12 13 14 15 16 17 33 18 19 20 21

Keywords: Familial hypercholesterolaemia Genetic therapy Antisense oligonucleotides RNA splicing Mipomersen Small interfering RNA PCSK9 Apolipoprotein B LDL receptor Low density lipoprotein

a b s t r a c t

45 46 47 48 49 50 51 52 53 54 55 56 57 58

Q4

C R

R

N C O

43 44

Familial hypercholesterolaemia (FH) is a common disease, characterised by high low density lipoprotein (LDL) cholesterol and premature atherosclerosis. Normally, the liver assembles very low density lipoprotein (VLDL) particles from the full-length isoform of Apolipoprotein B (APOB100) plus triglycerides, cholesterol and cholesteryl esters in the rough endoplasmic reticulum and the Golgi. After secretion, the VLDL particles are metabolised by peripheral tissue lipoprotein lipase into intermediate density and LDL particles (Fig. 1A). LDL particles are then cleared via the LDL receptor (LDLR) in the liver. FH is caused by mutations in LDLR, gain of function mutations in the serine protease PCSK9 which binds and designates LDLR for lysosomal degradation or accessory proteins such as LDLRAP1, which lead to loss-of-function of the LDLR. As a result, the normal clearance of LDL particles by binding of Apolipoprotein B100 (APOB100) to the LDLR on the cell surface of hepatocytes is disrupted, leading to accumulation of LDL particles in circulation which drive accelerated atherosclerosis (Fig. 1B). A similar physiological and clinical picture is observed in patients with familial defective APOB (FDB) who possess point mutations in the APOB gene that disrupt binding of APOB100 to the LDLR [1]. Heterozygous FH (HeFH) is the most common form, in which patients inherit one mutant allele of a causative gene, and is characterised

U

41 42

E

36

39 40

22 23 24 25 26 27 28 29 30 31 32

T

E

D

P

This review surveys the state-of-the-art in genetic therapies for familial hypercholesterolaemia (FH), caused most commonly by mutations in the LDL receptor (LDLR) gene. FH manifests as highly elevated low density lipoprotein (LDL) cholesterol levels and consequently accelerated atherosclerosis. Modern pharmacological therapies for FH are insufficiently efficacious to prevent premature cardiovascular disease, can cause significant adverse effects and can be expensive. Genetic therapies for FH have been mooted since the mid 1990s but gene replacement strategies using viral vectors have so far been unsuccessful. Other strategies involve knocking down the expression of Apolipoprotein B100 (APOB100) and the protease PCSK9 which designates LDLR for degradation. The antisense oligonucleotide mipomersen, which knocks down APOB100, is currently marketed (with restrictions) in the USA, but is not approved in Europe due to its adverse effects. To address this problem, we have devised a novel therapeutic concept, APO-skip, which is based on modulation of APOB splicing, and which has the potential to deliver a cost-effective, efficacious and safe therapy for FH. © 2014 Published by Elsevier Inc.

37 35 34

38

R O

i n f o

O

1

F

journal homepage: www.elsevier.com/locate/vph

⁎ Endocrinology, Division of Medicine, UCL, Royal Free Campus, London NW3 2PF, United Kingdom. Tel.: +44 20 77940500x33383. E-mail address: [email protected].

by elevations of LDL cholesterol N 4.9 mmol/L [2]. Homozygous FH (HoFH) is the most severe form in which patients inherit two mutant alleles, usually of LDLR. Similar severe HoFH clinical pictures are also seen when patients inherit two different mutant alleles of the same gene (compound heterozygosity) or two mutant alleles impacting different causative genes (e.g., LDLR plus one of either PCSK9, LDLRAP1 or APOB — double heterozygotes). HoFH is characterised by even higher LDL cholesterol levels N 13 mmol/L, and a first presentation of myocardial infarction typically in adolescence [3,4]. Although the prevalences of HeFH and HoFH in unselected populations have traditionally been quoted at 1 per 500 and 1 per million respectively, more recent data indicates that these conditions may be much more prevalent than previously recognised at 1 per 200 and 1 per 160,000–320,000 respectively [3]. FDB is also relatively common in the population at 1 per 1000 [5]. Frequently, patients with these conditions are not aware that they have hypercholesterolaemia until they present with myocardial infarction or stroke. This makes case detection and effective early treatment paramount in the management of these conditions. In HoFH patients, lipid lowering therapy, such as with high-dose, high-potency HmG CoA reductase inhibitors (‘statins’), is able to reduce the morbidity and mortality of atherosclerosis. However, present lipid lowering drugs are not potent enough to reduce LDL cholesterol levels to target. As a result, HoFH patients are still exposed to early development of cardiovascular disease, and they suffer side effects from the treatment such as myalgia and elevations in liver transaminases [6].

http://dx.doi.org/10.1016/j.vph.2014.12.002 1537-1891/© 2014 Published by Elsevier Inc.

Please cite this article as: Khoo B, Genetic therapies to lower cholesterol, Vascul. Pharmacol. (2014), http://dx.doi.org/10.1016/j.vph.2014.12.002

59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83

2

B. Khoo / Vascular Pharmacology xxx (2014) xxx–xxx

A

B liver

liver VLDL

VLDL LDL

LDL LPL

LPL

LDL IDL

LDL

IDL

LDL LDL

APOB100 LPL

LPL

F

LDLR

O

APOE

84 85

R O

Fig. 1. Physiology of VLDL secretion and processing into LDL (A) and the pathophysiology of FH (B). (A) The liver assembles VLDL particles containing triglycerides and cholesterol, APOB100 and Apolipoprotein E (APOE). The action of peripheral tissue lipoprotein lipase (LPL) hydrolyses the triglycerides to fatty acids which are taken up by the tissues, and the VLDL particles are progressively metabolised to intermediate density lipoprotein (IDL) and then low density lipoprotein (LDL). Some of the cholesterol-rich LDL particles are bound by LDLR and non-hepatic LDL receptors (scavenger receptors) in peripheral tissues, allowing cholesterol to be transported to these tissues. The remaining LDL particles (40–60%) are cleared by the liver via binding of LDL receptor (LDLR) to APOB100. (B) Defective or missing LDLR leads to reduced hepatic clearance of LDL particles, accumulation of LDL particles in circulation, and accelerated atherosclerosis.

123 124

93 Q6

1. LDLR gene replacement therapy

94

116

Given the fact that most cases of FH are due to defective LDLR, the simplest approach to therapy would be to transduce a good copy of LDLR into hepatocytes, restoring LDLR expression and function, and thereby reducing LDL cholesterol levels. Initially, promising results were obtained in animal studies, such as in Ldlr knockout mice where the introduction of the human LDLR gene via an adenoviral vector was shown to ameliorate the hypercholesterolaemia observed in these mice after high-cholesterol feeding [8]. It was therefore natural to move on to early clinical studies, and Grossman et al. were able to demonstrate successful transduction of LDLR into the livers of 5 patients with HoFH using a retroviral vector in 1995. In one patient, there was a significant reduction in LDL cholesterol, implying restoration of LDL catabolism [9]. Despite this significant early progress, the initial promise of gene replacement therapy has not been fulfilled. The main roadblocks to gene replacement have been in obtaining efficient transduction of enough copies of the LDLR gene into the liver to assure sufficient restoration of LDL clearance, and concerns regarding the safety of the viral vectors used for this purpose remain. More modern vectors such as adeno-associated virus (AAV) serotype 8 may address the efficiency issue, and show promise in animal models [10]. Even if successful, however, this LDLR replacement strategy does not address the subset of patients who have FH due to mutations in PCSK9, LDLRAP1, or those with FDB.

This approach has been adopted by Isis Pharmaceuticals/Genentech with their antisense oligonucleotide-based drug, mipomersen. Mipomersen is a hybrid antisense oligonucleotide (ASO) which combines 2′-O-methoxyethyl RNA ‘wings’ with a central portion of 10 nucleotides which is based on DNA. This ‘gap-mer’ ASO is able to bind to the APOB mRNA within hepatocytes. The hybridization of the central portion's DNA to the mRNA acts as a substrate for RNase H, which cleaves RNA/DNA hybrids. In this way, the APOB mRNA is destroyed, leading to successful knockdown of APOB100 expression and consequent reductions in LDL cholesterol [11,12]. In clinical trials in patients with HoFH, weekly subcutaneous injections of mipomersen were able to reduce LDL cholesterol levels by ~25% [13]. The expectation is that this will translate to reduced morbidity and mortality, supported by animal studies [14]. However, mipomersen's pathway to the bedside has been beset by safety issues. The most common adverse effects were injection site reactions, elevations in liver function tests and flu-like symptoms. As a result, mipomersen has only been approved by the FDA under a Risk Evaluation Mitigation Strategy (REMS) [15]. Mipomersen has not been approved by the European authorities who cited specific concerns regarding the adverse effects, the relatively high drop-out rate from treatment, and an apparent increase in cardiovascular events in treated patients, in contradiction to expectations [16]. Lastly, mipomersen has been marketed at a cost of at least US$176,000 per annum. Given that this is a lifelong treatment, the cost will limit its appeal and restrict its use only to the most affected HoFH patients. Small interfering RNA (siRNA) technology has also been used to knockdown APOB expression in non-human primates, leading to reductions of LDL cholesterol [17]. Because siRNA therapies require relatively unmodified RNA duplexes, they are generally formulated in delivery formulations to allow them to reach the liver without being degraded, in this case a lipid nanoparticle formulation called SNALP was used. This therapy, TKM-ApoB, underwent Phase I clinical trials in 2009, with LDL cholesterol reductions of 16.3% reported at the highest doses tested, but one subject had an immunological reaction to the drug [18]. Clinical development of this therapy appears to have been halted.

117

2. APOB100 knockdown

3. PCSK9 knockdown/inhibition

158

118 119

APOB100 is the integral structural apolipoprotein for LDL particles. As mentioned earlier, APOB100 is assembled with triglycerides, cholesteryl esters and cholesterol into VLDL particles by the liver before secretion. Therefore, down-regulation of APOB100 expression leads to reduced VLDL assembly and secretion, and reduced LDL particle levels.

PCSK9 is a protease of the subtilisin-like proprotein convertase family of enzymes which regulates the cell surface expression of LDLR by degradation [19]. As mentioned above, gain-of-function mutations in PCSK9 are responsible for some cases of FH. Logically, reduction/knockdown of PCSK9 will increase LDLR cell surface expression and will

159 160

101 102 103 104 105 106 107 108 109 110 111 112 113 114 115

120 121 122

D

E

T

C

E

R

99 100

R

97 98

O

95 96

C

90 91

N

88 89

U

86 87

P

92

Other treatments, such as LDL apheresis, are temporarily effective but are extremely expensive and invasive [7]. As a result, new treatments for HoFH are sorely needed. This review will summarise some recent developments in the field of therapy for FH. Specifically, it will concentrate on genetic approaches to FH therapy, notably APOB100 knockdown using antisense oligonucleotide and RNA interference approaches, PCSK9 knockdown to increase cell-surface LDLR expression, and splice-switching as a means of reengineering APOB expression and function.

Please cite this article as: Khoo B, Genetic therapies to lower cholesterol, Vascul. Pharmacol. (2014), http://dx.doi.org/10.1016/j.vph.2014.12.002

125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157

161 162 163

B. Khoo / Vascular Pharmacology xxx (2014) xxx–xxx

188 189 190 191 192

F

C

186 187

APOB100

E

184 185

APOB48

R

183

APOB100 mRNA

APOB

RNA editing

translation

R

181 182

N C O

175 176

Exon 26

27

28

29

APOB87 mRNA

U

173 174

O

A clue as to an entirely novel method for treating FH can be found in the condition familial hypobetalipoproteinaemia (FHBL). FHBL is due to heterozygous mutations in APOB that cause the expression of Cterminally truncated APOB100 isoforms such as APOB89 and APOB87Padova. The truncated APOB inhibits VLDL assembly and secretion, and LDL particles bearing APOB truncations are cleared faster from the circulation [24]. Patients with FHBL therefore have the antithetical phenotype to FH, with extremely low LDL cholesterol levels (typically b1.8 mmol/L). As a consequence, patients with FHBL are protected from cardiovascular disease and live longer than normal people [25]. Elective truncation of APOB100 would therefore be expected to replicate FHBL and to reduce LDL cholesterol. To accomplish this goal, we designed and tested a method to exclude exon 27 (‘exon-skipping’)

171 172

R O

179 180

170

P

4. APO-skip: splice-switching APOB

168 169

D

178

166 167

during RNA splicing, leading to a frameshift and premature termination codon within the exon 28 sequence, as opposed to the usual termination within exon 29 (Fig. 2). In turn, translation of the exon-skipped mRNA leads to the expression of a truncated APOB87 isoform [26]. This can be accomplished using APO-skip SSOs which are ASOs based on phosphorothioate 2′-O-methyl RNA chemistry. These are designed to bind to intronic splice sites flanking APOB exon 27, and confer a secondary structure to the pre-mRNA that favours exclusion of exon 27. With sequence optimization and chemical modification with phosphorothioate groups to confer stability to nucleases, exon-skipping efficiency has been increased up to 15-fold in vitro over the prototype SSOs tested in 2007 [28]. The APO-skip therapeutic concept was validated in hypercholesterolaemic mice transgenically expressing human APOB. APO-skip SSOs can durably reduce LDL cholesterol by 34–51% when injected at weekly intervals for 8 weeks. This is a striking result when it is realized that only 6% of the APOB mRNA has been exon-skipped, and the powerful reductions in LDL cholesterol achieved are due to the dual mechanism activated by the SSOs: reduced assembly plus increased clearance [28]. More recently we have developed 2nd and 3rd generation APO-skip SSOs that are, when combined with liver-specific delivery formulations, able to increase in vivo potency by over 10-fold. The prospect, therefore, is that powerful lipid lowering effects can be obtained with markedly smaller amounts of SSO, reducing costs and potential adverse effects. In addition, direct liver delivery is likely to obviate the development of skin reactions and anti-oligonucleotide antibodies which are observed in patients treated with therapeutic oligonucleotides such as mipomersen [15] (Fig. 3). The key difference that distinguishes the APO-skip technique from simple knockdown of APOB, as employed by mipomersen and TKMApoB, is that it does not interfere with intestinal fat transport and

T

177

reduce LDL levels by increasing hepatic clearance. Indeed, monoclonal antibodies against PCSK9 are capable of reducing LDL cholesterol levels in patients with HeFH by up to 68% [20]. Therapies based on siRNA to knockdown PCSK9 have also been shown to reduce LDL cholesterol in Phase I trials in healthy volunteers by 40% [21]. More recently, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nuclease genome editing has been used to knockdown PCSK9 expression in mice. This method, which involves vector-mediated delivery of ‘guide’ RNAs to mutagenise PCSK9 could potentially be developed into a ‘one-shot’ permanent solution to reduce LDL cholesterol [22]. However, the lipid lowering mechanism of PCSK9 knockdown is dependent on the presence of at least some functional LDLR. In patients with HoFH, PCSK9 inhibitors are markedly less effective and in some cases completely ineffective [23]. This limits the utility of this approach in HoFH.

E

164 165

3

RNA editing

translation

APOB48 APOB87

Fig. 2. How APOB exon 27 skipping leads to truncation of APOB87. The exons 26 to 29 at the 3′ end of the APOB gene are shown schematically as rectangles with the introns shown as connecting lines. In the liver, APOB100 is translated from the constitutively spliced APOB100 mRNA, which includes exon 27 (top). In the intestine, the shorter APOB48 isoform is translated from the APOB100 mRNA after RNA editing of a CAA to a UAA stop codon represented by the blue marker in exon 26 [27]. Exon-skipping exon 27 leads to the generation of APOB87 mRNA with frame-shifted exon 28 & 29 sequences and a premature stop codon (red marker in exon 28). Thus, a truncated APOB protein, APOB87, is translated. Exon 27 skipping does not interfere with the exon 26 sequence, so APOB48 RNA editing and expression can proceed normally. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Khoo B, Genetic therapies to lower cholesterol, Vascul. Pharmacol. (2014), http://dx.doi.org/10.1016/j.vph.2014.12.002

193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 Q7 221 222 223

B. Khoo / Vascular Pharmacology xxx (2014) xxx–xxx

40

20

30

0

ΔLDL (%)

exon 27 skipping %

4

20 10

-20 -40 -60

0 14-53P

sc1453P

UT

3w

statin

6w

8w

Time

8 x 20 mg/kg SSO+IVF2.0

249

256 257

Genetic therapies for FH have come a long way since the early trials of replacement gene therapy some 20 years ago. APOB100 knockdown has become a prime target for the treatment of FH and one genetic therapy, mipomersen, is currently marketed. However, the safety and tolerability issues that cloud mipomersen mean that new therapeutic concepts, preferably reducing adverse effects, are still needed. APOskip SSOs, which truncate APOB100 and lower LDL cholesterol via a unique dual mechanism, represent such a new concept with the potential to deliver a cost-effective, efficacious and safe therapy.

258

Acknowledgements

259 260

Support for the APO-skip project was given by the Medical Research Council Developmental Pathway Research Scheme (G0802469), and UCL Business. The author wishes to acknowledge and thank Dr. Petra Disterer, Dr. Paul Simons and Prof. James Owen for their crucial contributions to the APO-skip project. This article is based on a presentation at the 8th International Workshop on Cardiovascular Biology & Translational Medicine held in London in September 2013.

244 245 246

250 251 252 253 254 255

261 Q8 262 263 264 265

C

E

242 243

R

240 241

R

238 239

O

236 237

C

234 235

N

232 233

U

230 231

P

5. Conclusions

228 229

[1] Boren J, Ekstrom U, Agren B, Nilsson-Ehle P, Innerarity T. The molecular mechanism for the genetic disorder familial defective apolipoprotein B100. J Biol Chem 2001; 276(12):9214–8. [2] Marks D, Thorogood M, Neil HA, Humphries SE. A review on the diagnosis, natural history, and treatment of familial hypercholesterolaemia. Atherosclerosis 2003; 168(1):1–14. [3] Cuchel M, Bruckert E, Ginsberg HN, Raal FJ, Santos RD, Hegele RA, et al. Homozygous familial hypercholesterolaemia: new insights and guidance for clinicians to improve detection and clinical management. A position paper from the Consensus Panel on Familial Hypercholesterolaemia of the European Atherosclerosis Society. Eur Heart J 2014;35(32):2146–57. [4] Raal FJ, Pilcher GJ, Panz VR, van Deventer HE, Brice BC, Blom DJ, et al. Reduction in mortality in subjects with homozygous familial hypercholesterolemia associated with advances in lipid-lowering therapy. Circulation 2011;124(20):2202–7. [5] Tybjaerg-Hansen A, Steffensen R, Meinertz H, Schnohr P, Nordestgaard BG. Association of mutations in the apolipoprotein B gene with hypercholesterolemia and the risk of ischemic heart disease. N Engl J Med 1998;338(22):1577–84. [6] Marais AD, Raal FJ, Stein EA, Rader DJ, Blasetto J, Palmer M, et al. A dose-titration and comparative study of rosuvastatin and atorvastatin in patients with homozygous familial hypercholesterolaemia. Atherosclerosis 2008;197(1):400–6. [7] Thompson GR, Group H-ULAW. Recommendations for the use of LDL apheresis. Atherosclerosis 2008;198(2):247–55. [8] Ishibashi S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, Herz J. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery. J Clin Invest 1993;92(2):883–93. [9] Grossman M, Rader DJ, Muller DW, Kolansky DM, Kozarsky K, Clark III BJ, et al. A pilot study of ex vivo gene therapy for homozygous familial hypercholesterolaemia. Nat Med 1995;1(11):1148–54. [10] Kassim SH, Li H, Bell P, Somanathan S, Lagor W, Jacobs F, et al. Adeno-associated virus serotype 8 gene therapy leads to significant lowering of plasma cholesterol levels in humanized mouse models of homozygous and heterozygous familial hypercholesterolemia. Hum Gene Ther 2013;24(1):19–26. [11] Crooke R, Graham M, Lemonidis K, Whipple C, Koo S, Perera R. An apolipoprotein B antisense oligonucleotide lowers LDL cholesterol in hyperlipidemic mice without causing hepatic steatosis. J Lipid Res 2005;46(5):872–84. [12] Kastelein JJ, Wedel MK, Baker BF, Su J, Bradley JD, Yu RZ, et al. Potent reduction of apolipoprotein B and low-density lipoprotein cholesterol by short-term administration of an antisense inhibitor of apolipoprotein B. Circulation 2006;114(16):1729–35. [13] Raal FJ, Santos RD, Blom DJ, Marais AD, Charng MJ, Cromwell WC, et al. Mipomersen, an apolipoprotein B synthesis inhibitor, for lowering of LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia: a randomised, double-blind, placebo-controlled trial. Lancet 2010;375(9719):998–1006. [14] Mullick AE, Fu W, Graham MJ, Lee RG, Witchell D, Bell TA, et al. Antisense oligonucleotide reduction of apoB-ameliorated atherosclerosis in LDL receptor-deficient mice. J Lipid Res 2011;52(5):885–96. [15] Federal Drug Administration. NDA 203568: Kynamro (mipomersen sodium) Risk Evaluation and Mitigation Strategy 2014. Available from: http://www.fda.gov/ downloads/Drugs/DrugSafety/ PostmarketDrugSafetyInformationforPatientsandProviders/UCM337472.pdf. [16] European Medicines Agency. Refusal of the marketing authorisation for Kynamro (mipomersen) 2012. Available from: http://www.ema.europa.eu/docs/ en_GB/document_library/Summary_of_opinion_-_Initial_authorisation/human/ 002429/WC500136279.pdf. [17] Zimmermann T, Lee A, Akinc A, Bramlage B, Bumcrot D, Fedoruk M, et al. RNAimediated gene silencing in non-human primates. Nature 2006;441(7089):111–4. [18] Tekmira Pharmaceuticals Completes ApoB SNALP Phase 1 Clinical Trial [press release]; 2010. [19] Lambert G, Sjouke B, Choque B, Kastelein JJ, Hovingh GK. The PCSK9 decade. J Lipid Res 2012;53(12):2515–24.

D

248

226 227

References

T

247

metabolism. APOB48, the natural alternate isoform of APOB, is expressed exclusively in the intestine and is expressed by RNA editing of the APOB mRNA sequence within exon 26 to generate a premature termination codon (Fig. 2). As a result, APOB48, which is identical to the N-terminal 48% of the full-length liver APOB100 isoform, is expressed and assembled into the chylomicron particle, which transports dietary fat to peripheral tissues. Importantly, simple knockdown of the APOB mRNA causes an off-target downregulation of APOB48 expression and chylomicron levels as both APOB100 and APOB48 originate from the same mRNA. For example, siRNA knockdown causes a drop in chylomicron levels of 50% [17]. This could lead to fat malabsorption, steatorrhoea, malabsorption of fat-soluble vitamins such as Vitamin K, and therefore potential interactions with drugs such as warfarin. Similar adverse effects are observed in clinical trials with the microsomal triglyceride transfer protein inhibitor lomitapide, which also interferes with chylomicron assembly [29]. Because APO-skip SSOs do not reduce APOB48 expression [28] this off-target effect is obviated, again reducing the potential for adverse effects. Mipomersen, lomitapide and APO-skip SSOs share a common mechanism in reducing VLDL assembly in the liver and this is expected to cause fat accumulation in the liver, similar to that observed in patients with FHBL. The clinical significance of this hepatosteatosis is unclear; in patients with FHBL it does not seem to be associated with other metabolic derangements such as insulin resistance [30].

E

224 225

R O

O

F

Fig. 3. APO-skip SSOs reduce LDL cholesterol in vivo. APO-skip SSO 14-53P and scrambled negative control sc14-53P were formulated in Invivofectamine 2.0 and injected at 20 mg/kg on a weekly basis into transgenic human APOB (hAPOB) mice. Two other groups of mice were given pravastatin (statin) and ordinary tap water (untreated, UT). Left panel: At termination, total liver RNA was extracted and analysed for exon 27 skipping: mean 7 ± S.D. 4% with SSO 14-53P, no significant skipping detected in other groups. Right panel: Percentage changes in LDL cholesterol corrected for changes observed for the placebo groups: week 3 SSO 14-53P (green; −51 ± 19%) and Pravastatin (grey; −13 ± 13%), week 6 SSO 14-53P (−33 ± 23%) and Pravastatin (−4 ± 6%) and week 8 SSO 14-53P (−34 ± 12%) and Pravastatin (−3 ± 9%). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Khoo B, Genetic therapies to lower cholesterol, Vascul. Pharmacol. (2014), http://dx.doi.org/10.1016/j.vph.2014.12.002

266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325

B. Khoo / Vascular Pharmacology xxx (2014) xxx–xxx

326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342

[20] Stein EA, Gipe D, Bergeron J, Gaudet D, Weiss R, Dufour R, et al. Effect of a monoclonal antibody to PCSK9, REGN727/SAR236553, to reduce low-density lipoprotein cholesterol in patients with heterozygous familial hypercholesterolaemia on stable statin dose with or without ezetimibe therapy: a phase 2 randomised controlled trial. Lancet 2012;380(9836):29–36. [21] Fitzgerald K, Frank-Kamenetsky M, Shulga-Morskaya S, Liebow A, Bettencourt BR, Sutherland JE, et al. Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blind, placebo-controlled, phase 1 trial. Lancet 2014;383(9911):60–8. [22] Ding Q, Strong A, Patel KM, Ng SL, Gosis BS, Regan SN, et al. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ Res 2014;115(5):488–92. [23] Stein EA, Honarpour N, Wasserman SM, Xu F, Scott R, Raal FJ. Effect of the proprotein convertase subtilisin/kexin 9 monoclonal antibody, AMG 145, in homozygous familial hypercholesterolemia. Circulation 2013;128(19):2113–20. [24] Schonfeld G. Familial hypobetalipoproteinemia: a review. J Lipid Res 2003;44(5): 878–83.

5

[25] Glueck CJ, Gartside PS, Mellies MJ, Steiner PM. Familial hypobeta-lipoproteinemia: studies in 13 kindreds. Trans Assoc Am Physicians 1977;90:184–203. [26] Khoo B, Roca X, Chew SL, Krainer AR. Antisense oligonucleotide-induced alternative splicing of the APOB mRNA generates a novel isoform of APOB. BMC Mol Biol 2007; 8:3. [27] Chester A, Scott J, Anant S, Navaratnam N. RNA editing: cytidine to uridine conversion in apolipoprotein B mRNA. Biochim Biophys Acta 2000;1494(1–2):1–13. [28] Disterer P, Al-Shawi R, Ellmerich S, Waddington SN, Owen JS, Simons JP, et al. Exon skipping of hepatic APOB pre-mRNA with splice-switching oligonucleotides reduces LDL cholesterol in vivo. Mol Ther 2013;21(3):602–9. [29] Cuchel M, Meagher EA, du Toit Theron H, Blom DJ, Marais AD, Hegele RA, et al. Efficacy and safety of a microsomal triglyceride transfer protein inhibitor in patients with homozygous familial hypercholesterolaemia: a single-arm, open-label, phase 3 study. Lancet 2013;381(9860):40–6. [30] Visser ME, Lammers NM, Nederveen AJ, van der Graaf M, Heerschap A, Ackermans MT, et al. Hepatic steatosis does not cause insulin resistance in people with familial hypobetalipoproteinaemia. Diabetologia 2011;54(8):2113–21.

U

N C O

R

R

E

C

T

E

D

P

R O

O

F

360

Please cite this article as: Khoo B, Genetic therapies to lower cholesterol, Vascul. Pharmacol. (2014), http://dx.doi.org/10.1016/j.vph.2014.12.002

343 344 345 346 347 348 349 350 351 352 353 Q9 354 355 356 357 358 359